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Messenger RNA for thyroid hormone receptor (TR) isoforms alpha 1 and beta 1 are widely distributed in rat tissues. Until recently, TR beta 2 mRNA was believed to be limited to the pituitary and the assumption was made that TR beta 2 protein was similarly restricted. We determined the distribution of TR beta 2 protein in selected adult and fetal rat tissues using three anti-TR beta 2 antisera directed to different amino acid sequences of the distinctive A/B domain of TR beta 2. The proportion of total nuclear binding capacity cleared by each antiserum was determined by saturation analysis. 10-20% of total binding capacity in adult brain, liver, kidney, and heart was immunoprecipitated by each antiserum. Use of specific antibodies to TR beta 1 and TR alpha 1 showed these isoforms accounted for the remainder of total T3 binding. Fetal liver and brain, however, contained only TR alpha 1. Immunohistochemical analysis of the adult tissues showed TR beta 2 present in nuclei. Reverse transcription polymerase chain reaction detected low levels of TR beta 2 mRNA in the adult tissues. We infer that TR beta 2 accounts for a significant fraction of TR in adult rat tissues despite the low levels of its mRNA.

We have determined the contribution of the thyroid hormone receptor (TR) isoforms TR alpha 1 and TR beta 1 to the postnatal rise in rat hepatic nuclear T3-binding capacity. In agreement with previous studies, total hepatic nuclear binding capacity rose by about 8-fold from the 19th day of gestation to young adulthood at 2 months of age (0.10 +/- 0.03 to 0.86 +/- 0.17 pmol/mg DNA). The levels of specific TR species were measured by immunoprecipitation of T3-binding activity from hepatic extracts using a panel of antisera directed against specific regions of the TR isoforms. The difference between receptor immunoprecipitated with antibody against TR beta 1 and that precipitated with an antibody against an identical region in both TR beta 1 and TR alpha 1 was tentatively assumed to represent TR alpha 1. TR alpha 1 accounted for virtually all T3-binding activity in fetal liver on gestational day 19 (G19), increased by 2-fold shortly after birth, and remained constant thereafter. TR alpha 1 mRNA, on the other hand, was highest in concentration on G16 and fell by 50-75% in the adult. TR beta 1 was undetectable by immunoprecipitation of hepatic extracts from fetuses on G19. However, Northern analysis showed the presence of TR beta 1 mRNA in the fetal liver, which rose in concentration by 3- to 4-fold in late gestation and then remained constant. The contribution of TR beta 1 to total binding capacity rose to 33% and 40% on postnatal days 15 and 30, respectively, and to 80% in the adult liver. Immunohistochemical analyses of hepatic sections confirmed the presence of very low levels of TR beta 1 in fetal liver as early as G16 and G19, and a sharp rise in TR beta 1 protein concentration in the postnatal period. This indicated that the increase in TR beta 1-binding capacity results from increased TR beta 1 mass. The increase in TR beta 1-binding capacity, thus, is due to increased translational efficiency of the beta 1 mRNA or stabilization of the TR beta 1 protein. The prominence of TR alpha 1 in both rat fetal liver and fetal brain, as previously demonstrated in our laboratory, raises the possibility that this receptor isoform may carry out specialized functions in the fetus and that TR beta 1 subserves still other functions at later stages of development.(ABSTRACT TRUNCATED AT 400 WORDS)

A panel of anti-thyroid hormone receptor (TR) antisera were generated to allow direct assay of the concentrations of the alpha 1 and beta 1 receptor isoforms in nuclear extracts from adult rat liver, kidney, brain and heart, and fetal brain. An antiserum, immunoglobulin G (IgG)-beta 1, raised against amino acid sequence 62-92 of the rat TR-beta 1 specifically precipitated only TR-beta 1 in vitro translation products. A second antiserum, IgG-alpha 1/beta, generated against a sequence that is identical in the ligand binding region of rat TR-alpha 1 and TR-beta isoforms immunoprecipitated both TR-alpha 1 and -beta 1 translation products. These IgG preparations were used to specifically immunoprecipitate thyroid hormone receptor binding activity from nuclear extracts. IgG-beta 1 cleared almost 80%, and the IgG-alpha 1/beta immunoprecipitated nearly all binding from hepatic nuclear extracts. This distribution of TR protein, 80% beta 1 and 20% alpha 1, is the same as previously reported for their respective mRNAs in liver. In heart, kidney, and brain IgG-beta 1 cleared 45, 43, and 28% of total binding, respectively, and IgG-alpha 1/beta cleared all T3 binding activity from these tissues. In agreement with an earlier study, marked variations in specific protein/mRNA ratios were noted among these tissues. Consistent with our earlier report of the presence of only very low levels of TR-beta 1 mRNA in fetal brain, IgG-beta 1 cleared just 5% of binding in this tissue. Studies using an antiserum (IgG-ch) generated against homologous segments of the hinge region in both TR-alpha 1 and -beta 1 yielded results which contrasted sharply with those of IgG-alpha 1/beta. Whereas IgG-ch could also immunoprecipitate virtually all binding from hepatic extracts it cleared only 40-50% of binding from the other tissues, including fetal brain in which TR-alpha 1 accounts for greater than 90% of binding protein. The data suggest the presence of posttranslational modification of the TR-alpha 1 protein in the hinge region, consistent with the presence in this segment of potential phosphorylation sites.

We have quantitated in adult and developing rat tissues the molar concentrations of c-erbA alpha 1- and beta 1-mRNAs, which code for nuclear T3-binding proteins, and c-erbA alpha 2-mRNA, which is generated by alternate splicing of the alpha gene transcript and codes for a receptor variant that does not bind T3. Comparison of the concentrations of c-erbA alpha 1-mRNA, beta 1-mRNA, or their sum to the T3 nuclear binding capacity per mg of DNA in adult liver, kidney, heart, cerebrum, and cerebellum and during the ontogeny of liver and brain shows that the T3 binding capacity/c-erbA mRNA ratio is tissue-specific and related to developmental state. Administration of T3 resulted in a 40-50% fall in the alpha 1 signal of adult liver, kidney, and heart without changing either the beta 1 signal or T3 binding capacity. A 40-fold increase in rat brain beta 1-mRNA occurred in the transition between the 19-day gestational fetus and the 10-day-old neonate. This corresponds to the period during which the T3 content rises in brain and during which T3 is known to influence central nervous system development. Our findings indicate that important translational or post-translational factors influence nuclear binding capacity and raise the possibility that c-erbA beta 1 may play a primary role in mediating T3 effects in developing and adult animals.

We have investigated the thermodynamic properties of triiodothyronine (T3)-solubilized hepatic nuclear receptor interaction. These studies revealed that the equilibrium constant was markedly dependent on temperature and van't Hoff plots revealed a significant deviation from linearity. Both enthalpy and entropy changes decreased with increasing temperature such that at 37 degrees C both were negative. (delta H0 -27.1 kcal/mol; delta S0, -45.1 cal/mol.deg; and the heat capacity delta Cp0 (25 degrees C), -759 cal/mol.deg). Several T3 analogues revealed similar thermodynamic characteristics. Our finding that the T3-receptor interaction is characterized by a negative heat capacity is compatible with the previous proposal of hydrophobic bonding by Jorgensen. The large negative entropy change at 37 degrees C for T3-receptor reaction is in contrast to the more positive entropy values for several T3-plasma protein reactions. One possible explanation of this difference in entropy values between the nuclear receptor and plasma proteins is that T3 induces a conformational change in the receptor, a concept supported by previous data from our laboratory indicating that the occupation of the T3 receptor results in an alteration in the chromatographic migration of the receptor.

Further studies have been performed to define the kinetic characteristics of nuclear triiodothyronine (T(3)) binding sites in rat liver (J. Clin. Endocrinol. Metab. 1972. 35: 330). Sequential determination of labeled T(3) associated with nuclei and cytoplasm over a 4-h period allowed analysis of the relationship of T(3) in nuclear and cytoplasmic compartments. A rapid interchange of hormone between nuclei and cytoplasm was demonstrated, and in vitro incubation experiments with nuclei yielded no evidence favoring metabolic transformation of T(3) by the nuclei. In vivo displacement experiments were performed by subcellular fractionation of liver (1/2) h after injection of [(125)I]T(3) with increasing quantities of unlabeled T(3). The nuclear binding capacity for T(3) could be defined (0.52 ng/mg DNA). Analysis of these experiments also allowed an estimation of the association constant of nuclear sites for T(3) (4.7 x 10(11)M(-1)). The affinity of these sites for T(3) was estimated to be 20-40 fold greater than for thyroxine (T(4)). Chromatographic analysis of the nuclear radioactivity after injection of labeled T(4) indicated that the binding of T(4) by the nucleus could not be attributed to in vivo conversion of T(4) to T(3) but reflected intrinsic cross-reactivity of the two iodothyronines at the nuclear binding sites.

The limited capacity, high affinity binding of 35 iodothyronine analogues by rat liver nuclei has been examined in an in vitro system. The in vitro nuclear binding of all the analogues tested was highly correlated with their published thyromimetic potencies in the intact animals. Binding and biological activity are greater for 3'-mono-than 3',5'-di-substituted iodothyronines. A 4'-hydroxyl group is essential, but the 3' substituent can be several halogen or non-halogen groups for which the distal conformation is preferred. The ether linkage can be replaced equally well by a methylene or sulfur group. The presence of both 3 and 5 groups which are limited to halogens or small alkyl groups are necessary for the maintenance of significant activity. Halogen-free iodothyronines have very low, but significant activity both in vitro and in vivo. The data provide information on the structural requirements for thyroid hormone action and further support the physiological relevance of the nuclear sites.

We have investigated the transport of L- and D-triiodothyronine (T3) from plasma to cellular cytoplasm and from cytoplasm to nucleus by estimating the concentration of free hormone in these compartments in rat liver, kidney, brain, and heart. We assessed the distribution of T3 in various tissues and its metabolism by standard isotopic techniques and measured plasma and cytosolic tissue T3 by radioimmunoassay. In addition, we determined the fraction of radiosensitive T3 associated with the cytosol in individual tissues and estimated the cytosolic volume per gram of tissue. Equilibrium dialysis allowed us to determine the binding power of cytosols and plasma, and in vitro saturation techniques provided values for the affinity (ka) for L- and D-T3 of isolated nuclei in aqueous solution at 37 degrees C. We calculated the free cytosolic hormone from the product of cytosolic T3 and the binding power of cytosol for T3, and the free intranuclear T3 from the ka and previously determined ratio of occupied-to-unoccupied binding sites under steady state conditions in euthyroid animals. Our results showed that the free cytosolic/free plasma concentrations for L-T3 and D-T3, respectively, were: liver 2.8, 21.6; kidney 1.17, 63.3; heart 1.31, 1.58; brain 0.86, 0.24. The free nuclear/free cytosolic ratios for L-T3 and D-T3, respectively, were: liver 58.2, 3.70; kidney 55.9, 1.54; heart 80.6, 24.9; and brain 251, 108.6. Our findings suggest that stereospecific transport occurs both from plasma to cytosol and from cytosol to nucleus. The high gradients from cytosol to nucleus imply that there is an energy-dependent process and appear to account for the differences in the nuclear association constant determined in vivo and in vitro.

Recent findings have led to a greater understanding of thyroid hormone action. The nuclear receptor for triiodothyronine is an integral component of a larger chromatin fragment. A stereospecific energy-dependent transport system appears responsible for translocation of triiodothyronine from cytosol to nucleus. In the liver, a multiplicative interaction between a signal from the triiodothyronine-nuclear receptor complex and a signal generated from carbohydrate metabolism results in the induction of specific mRNAs. Two-dimensional mRNA activity profiles suggest that approximately 8% of the visible mRNA sequences are differentially affected by alterations in thyroid states. Almost 30% to 40% of these changes are mediated by an increase in pituitary growth hormone induced by triiodothyronine. Sequential analyses of mRNA activity profiles have identified an mRNA sequence (mRNAs14) coding for a protein (S14) with Mr 17 010 and pI 4.9 which responds to triiodothyronine with a lag time of less than 20 minutes. The coordinate regulation of mRNAs14 by carbohydrate and triiodothyronine and its presence in lipogenic tissues (fat, liver, lactating mammary tissue) suggests that S14 is involved in some aspect of fatty acid synthesis degradation or storage.

In vivo saturation studies of thyroidectomized, propyl thiouracil, and L-triiodothyronine treated animals indicate that the hepatic nuclear binding capacity is not significantly influenced by the thyroidal status of the tissues. The constancy of the nuclear binding capacity for L-triiodothyronine facilitated an analysis of the relationship between nuclear occupancy and hepatic response to an intravenous injection of a triiodothyronine pulse. The activity of the mitochondrial enzyme alpha-glycerophosphate dehydrogenase was used as an index of tissue response to the administered triiodothyronine. Results were compatible with the following model. Saturation of nuclear sites is rapidly followed by a maximal rate of enzyme synthesis. When the sites are saturated, the activity of alpha-glycerophosphate dehydrogenase appeared to be independent of the dose administered. Higher doses of triiodothyronine achieve greater effects simply by occupying nuclear sites for a longer period. The apparent ability of nuclear sites to constrain tissue response to hormone administration provides additional supporting data for the physiological relevance of these sites in the initiation of hormonal action.

We have recently demonstrated substantial stereospecific nuclear/cytosolic free triiodothyronine (T3) gradients within T3 responsive rat tissues in situ. These studies have now been extended to examine T3 transport in a rat pituitary tumor cell line, GH1. L-T3 had a 7.6-fold higher affinity for the nuclear receptor when assayed in whole cell incubations in comparison to isolated nuclei, though D-T3 affinity was not altered under these conditions. An apparently higher number of receptors for D-T3 was explained by racemic contamination of the isotopes used. Measurement of free hormone concentration ratios for both enantiomers revealed a small step up from medium to cytosol for L-T3 (1.65) but a reverse ratio for D-T3 (0.46). The nuclei were able to concentrate both enantiomers, though stereospecificity was maintained (nucleus/cytosol, L-T3, 4.5, D-T3 1.7). Transport of L-T3 at both boundaries could be inhibited by monodansylcadaverine. Thus, stereospecific transport functions are found within GH1 cells, though the magnitude of the free nucleus/cytosol gradient is reduced from those seen in rat tissues in situ.

Isolated hepatic nuclei from euthyroid rats were incubated with tracer (125I)L-triiodothyronine (T3) and increasing doses of nonradioactive T3 for 30 min at 37degrees C. The T3 bound specifically to nuclear sites increased with increasing T3 doses to a plateau, which represented the nuclear binding capacity, M. Addition of 1 mM KCN, NaF, dinitrophenol, oriodoacetate did not affect nuclear binding, indicating that active metabolism was not required. Kinetic studies showed that the nuclear sites were equilibrated with T3 within 30 min of incubation (one-half maximal binding at 3 min) and that the rate of release of T3 in vitro (0.058 min-1) was the same for endogenous T3 or for T3 bound to nuclei in vitro. Nuclear T3 resisted extraction with 0.14 M NaC1 buffered at pH 7.5, but both endogenous hormone and T3 bound in vitro were readily extracted by 0.4 M KC1 at pH 8.0. The elution profiles of endogenous and in vitro-bound T3 from Sephadex G-100 columns showed a common protein peak with a molecular weight of 60-65,000, assuming a globular protein. Scatchard analysis of in vitro displacement studies showed a single class of binding sites. Mean M equals 0.23 times 10-9 M or 0.85 ng T3 for nuclei isolated from 1 g of liver. Mean M closely corresponded to that anticipated from reported in vivo studies. The apparent association constant Ka for the nuclear sites, 5.55 times 108 M-1, was lower than in studies in vivo, probably attributable to the different ionic milieu of nuclei in the incubation buffer and in the intact cell. Thus, the identity of the nuclear T3 binding sites studied in vitro to those reported for endogenous hormone is demonstrated by similar binding capacities, release rates, analogue binding affinities (previously reported), and localization to chromatin nonhistone proteins of comparable molecular weight. The role of cytosol protein in nuclear binding was assessed by comparing binding parameters for extensively washed nuclei and nuclei incubated either with contaminating or added cytosol. No difference in Ka or M was found. Moreover, it was unlikely that specific cytosol proteins were already present in nuclei and functioned during incubation as a shuttle for T3, since Ka and M for nuclei obtained from athyreotic rats were similar to Ka and M for nuclei from euthyroid animals. Thus, an initial interaction between T3 and specific cytosol proteins does not appear to be a prerequisite for translocation of T3 to nuclear sites.

A large body of circumstantial evidence indicates that receptors located in nuclei of T3 responsive tissues represent a site of initiation of thyroid hormone action at the cellular level. Partial characterization of T3 receptors indicates that these proteins are monomeric structures in nuclei and are chromatin-associated non-histone proteins. Treatment of rat liver nuclei with either pancreatic DNase I or micrococcal nuclease releases T3 receptors from nuclei in two forms: a predominant (95 400 Mr; 5.5-6.0S) and a minor (265 000-365 000 Mr; 12.5S) nucleoprotein complex. Similar structures are excised from rat kidney, brain, and heart nuclei and from GH1 pituitary cell nuclei by micrococcal nuclease digestion. These endonuclease-excised receptor-containing complexes are significantly larger than the salt-extracted receptor (50 000 Mr; 3.5S). The presence of DNA and other non-receptor proteins in these structures indicates that T3 receptors probably function within multimeric complexes in vivo. Although T3 receptors appear to be associated with DNA between nucleosomes, i.e. linker DNA, it is not entirely clear whether all or only a fraction of T3 receptors interact with nucleosomal components. The 12.5S receptor-containing nucleoprotein complex may represent T3 receptors in association with linker DNA and nucleosomal components. T3 receptors do not appear to be uniformly distributed to all chromatin fractions, but are associated with structures having characteristics of transcriptionally active chromatin. They are found in a region of chromatin which is enriched in RNA polymerase activity, rapidly labeled RNA and non-histone proteins, and depleted of histone Hl. This region is also highly sensitive to both micrococcal nuclease and pancreatic DNase I digestion. The association of receptors with transcriptionally active chromatin, however, must be considered provisional until additional details of the precise receptor-chromatin interaction have been established. The recent demonstration of a 20-fold increase in a specific hepatic mRNA four hours following administration of T3 to hypothyroid rats indicates that thyroid hormone potentially has very rapid effects on hepatic gene expression. However, significant changes in nuclear protein phosphorylation, nuclear protein composition, and chromatin structure have not been detected within this four-hour period. Thus, effects of T3 on hepatic gene expression are brought about by local and presumably subtle changes in nuclear function.

We have studied the interrelationship of plasma and hepatic nuclear T3 in two lower vertebrates, lake trout and sea lamprey. Specific nuclear T3-binding sites were observed in liver of both species. Binding affinities for T3 and several analogs in lamprey and trout were similar to those in rat liver. The findings of identical sedimentation coefficients, Stokes radii, and mol wt suggest a high degree of homology of the receptor molecule among these species. In lamprey ammocoete, both total (approximately 26 nM) and free (approximately 0.12 nM) T3 concentrations were 7- to 36-fold higher than in the trout or rat. The concentration of free T4 was 4 times greater than that in rat plasma despite somewhat lower total T4 levels. This resulted from a 6 times lesser binding of T4 by ammocoete plasma. Total and free T3 and T4 levels fell sharply during transformation to the adult form. In trout, a 27 times greater concentration of free T3 in hepatic nuclei than in cytosol, previously documented for rat liver, gave evidence of the early evolution of an active transport process between these compartments. Analysis of [125I]T3 interchange in trout indicated the existence of two kinetically distinct compartments within the liver, an outer compartment in rapid equilibrium with plasma, and a slowly equilibrating compartment decaying with a t1/2 of 3.7 h. T3 associated with the nuclear receptor appeared to be in equilibrium with the slower hepatic compartment. Despite the apparent resemblance of T3 receptors in these species to those in the rat, administration of T3 (200 micrograms/100 g BW for 4 days) to trout caused no increase in hepatic oxygen consumption or the activities of malic enzyme or alpha-glycerophosphate dehydrogenase. Further, such treatment produced no consistent changes in hepatic mRNA activity profiles analyzed by two-dimensional electrophoresis of in vitro translational products. As suggested by the high free T3 concentration in the larval lamprey ammocoete, T3 in early vertebrates may have a primarily developmental function, manifested over a restricted time frame.